Immunology Summit, 2015
Dr. Simon J. Clark MRC Career Development Fellow Centre for Ophthalmology and Vision Sciences University of Manchester

Immunology Summit, 2015
Age-related macular degeneration (AMD)
• Largest cause of blindness in developed world. ~50 million people affected worldwide • • Occurs around 50 y.o. - Incidence will only ever increase with burgeoning elderly population Progressive destruction of the retina, preceded by hallmark lesions (drusen), leading to central vision loss • Anti-VEGF treatment for ‘wet’ AMD, only halts damage already suffered • No effective treatments for ‘dry’ AMD Black and Clark (2015) Genet. Med. online 28 th May Healthy Choroidal Neovascularisation Early AMD (drusen) Geographic atrophy

Immunology Summit, 2015
AMD – risk factors
• Risk factors include environment (i.e. diet, smoking) but is essentially a genetically driven disease

Immunology Summit, 2015
Alternative pathway of complement activation
McHarg S et al. (2015) Mol. Immunol. 67, 43-50.

Immunology Summit, 2015
The alternative pathway of complement
C3b
Activator (non-host) surface
C3b FB C3bBb C3 C3b C3b
Complement factor H
C3 b
- polyanions
FI C3b
activator (host) surface
C3b C3bBb C3b C3b C3b C3b C3b C3b C3b iC3b Inflammation
Phagocytosis MAC formation MAC formation Phagocytosis

AMD – risk factors
Immunology Summit, 2015

Immunology Summit, 2015
Recent discovery about the role of FHL-1
• Recently identified that Factor H-like protein 1 (FHL-1) concentrates on Bruch’s membrane • Still able to regulate complement but to date no one has worried about elucidating the level of involvement in immune regulation RPE Bruch’s membrane Choroid Green = FHL-1 Red = FH Clark S.J. et al. (2014) J. Immunol. 193, 4962-4970.

The role of FHL-1
Immunology Summit, 2015
Green = FHL-1 Red = FH

Immunology Summit, 2015
Curiously, FH seems to coat drusen
• The FH that is secreted by the RPE cells ‘coats’ drusen that form in Bruch’s membrane FHL-1 Clark S.J. et al. (2014) J. Immunol. 193, 4962-4970.

Immunology Summit, 2015
FHL-1 is locally expressed by RPE cells
• First to demonstrate that FHL-1 is locally expressed by the RPE cells in human eyes C3 CFB CFI FHL-1 CFH Best-1 RPE65 B-actin TBP 5 4 3 2 1 0 FHL1 CFH C3 CFB CFI Clark S.J. et al. (2014) J. Immunol. 193, 4962-4970.

Immunology Summit, 2015
FHL-1 can diffuse through Bruch’s (FH can not)
• First to demonstrate that FHL 1 can passively diffuse through Bruch’s membrane, and that FH can not • Noted that virtually no AMD associated form of FHL-1 (402H) was retained by the Bruch’s membrane – i.e. not being recruited, therefore not able to protect against complement attack Clark S.J. et al. (2014) J. Immunol. 193, 4962-4970.

Immunology Summit, 2015
What about the factor H-related (FHR) proteins?
• One major problem with investigating complement in AMD – it was still virtually impossible to distinguish between FH, FHL-1 and the 5 FHR proteins in tissue

Immunology Summit, 2015
Targeted mass spectrometry
• We have identified an enzyme that will cleave FH, FHL-1 and FHR1-5 and produce (at least one) unique peptide(s) identifiable by mass spectrometry. • Selected Reaction Monitoring MS (SRM-MS) detects peptides using a defined (calculated from peptide sequence).
m/z 673.2
m/z 624.2
Detector Q1 Q3 • We have had each of thee synthesized and used them to develop targeted mass spectrometry methodology that can detect all 7 proteins in tissue – Begun work on Bruch’s membrane – Radiolabeling our standard peptides will make technique quantifiable

Immunology Summit, 2015
Therapeutic potential
• Complement is very much a focus for novel therapeutic strategies to address AMD • A number of complement modulating therapies currently in various stages of clinical trial with a range of results Black and Clark (2015) Genet. Med. online 28 th May

Immunology Summit, 2015
Manchester Eye Tissue Repository (ETR)
• To support eye research in Manchester we generated a unique resource of human eye tissues • Manchester Royal Eye Hospital Eye Bank – 2000 pairs of eyes per year for corneal transplantation service – Remaining globes now given to ETR • Collect medical record data, phenotype for retinal dystrophies, genotyped (AMD only), dissect and store virtually all aspects of the eye: – Sclera; optic nerve; vitreous; neurosensory retina; Bruch’s membrane; choroid; remaining DNA from genotyping, culturing RPE cells – Also macular and peripheral biopsy punches for IHC

Immunology Summit, 2015
Manchester Eye Tissue Repository (ETR)
GA Drusen • Hoping to open the facility to other researchers who are working on ocular disease

Acknowledgements
Immunology Summit, 2015

Immunology Summit, 2015
Bruch’s membrane
• Bruch’s membrane is an acellular extracellular matrix (ECM) that supports the retinal pigment epithelium (RPE) cells, and subsequently the neurosensory retina.
• Bruch’s membrane helps filter nutrients from the choroid that help nourish the RPC cells, but also helps keep out unwanted material/cells.
Clark and Bishop (2015) J. Clin. Med. 4, 18-31

Immunology Summit, 2015
Enrichment of Bruch’s membrane
• Publishing detailed method of how to enrich Bruch’s membrane • Can remove RPE, choroid and contaminating blood proteins McHarg S et al. J. Vis. Exp. In press

Immunology Summit, 2015
Complement and Bruch’s membrane
• Bruch’s membrane is one of only two ‘basement membrane’ ECM directly in contact with the body’s blood flow, without prior contact with cell barriers or tight junctions – The other is the glomeruli basement membrane (GBM) in the kidney
Glomeruli RPE Bruch’s membrane Choroid Bowman’s capsule